How big of a problem is the issue of host-cell protein and DNA in biologic products?

Dr. Kibbey: Host cells, or cell substrates such as E. coli, yeast cells, or mammalian cells like Chinese hamster ovary (CHO), are used to express proteins that ultimately become recombinant therapeutic products. Residual host-cell proteins (HCP) and DNA are process impurities that remain in a recombinant drug product following purification.

The FDA requires manufacturers to report how much residual HCP or DNA from the host cell remains in the drug substance after purification. Levels of these materials are also monitored during process development and validation, and help manufacturers select the best methods to reduce these impurities.

In the case of residual DNA, validation that the purification process reduces residual DNA to acceptable levels (usually 100 pg or less in biopharmaceuticals but higher levels are sometimes allowed) may reduce or eliminate the need for residual DNA measurements for lot release.

Because recombinant therapies are themselves proteins, detection of residual host-cell DNA against the backdrop of proteins is relatively more straightforward than the detection of residual HCP. In contrast, many different types of proteins are expressed by a host cell, and they occur over a wide range of concentrations.

Thus, residual HCP measurement methods must be able to sensitively identify multiple proteins in the presence of other high-abundance proteins and drug product (often 1–100 ng HCP/mg drug product or ppm).

What is the impact of host-cell impurities on clinical safety and efficacy?

Dr. Kibbey: During the course of production of a therapeutic protein, host-cell DNA may remain in the final product. Host-cell DNA is often fragmented into many pieces as it is processed, and it is difficult to determine what, if any, clinical concerns would be posed by these DNA segments.

A potential concern associated with residual host-cell DNA is the transmission of oncogenes that could eventually lead to tumor formation in the patient receiving the therapy.

With regard to host-cell proteins, immunogenicity is a potential concern. Antibodies may form against the therapeutic protein itself, or may even form against endogenous proteins that are homologous or identical to host-cell proteins. Concerns associated with immunogenicity also may be related to the state of a patient receiving a therapy (e.g., persons working in laboratories may already have pre-existing levels of antibodies present in their systems due to exposure to mice).

Host-cell impurities also may impact the efficacy of a final product and/or be toxic. Thus, detection and measurement of host-cell impurities must be addressed appropriately.

What are some current techniques for testing for host-cell impurities and what are their advantages?

Dr. Kibbey: Technologies used to detect residual HCP differ from those used to test for residual DNA. While several methods can be applied for host-cell DNA detection, PCR is one of the most robust used by industry today. In the case of host-cell proteins, immunological assays (e.g., ELISA) in animals are often used for protein detection.

However, given that a broad spectrum of proteins can be present in a final therapeutic protein product, being able to detect host-cell proteins amid a complex population of proteins can be a challenge. While antibody-based assays are sensitive, antibodies will only form against the presence of proteins that are immunogenic for animals used to produce the antibodies for the immunological assay.

Please describe some novel technologies on the horizon that appear promising for host-cell impurity testing.

Dr. Kibbey: As previously mentioned, not all HCP found in a final therapeutic product is going to be immunoreactive or detected in antibody-based assays, especially given the large number and variety of proteins present. Thus, orthogonal methods for detection (e.g., mass spectrometry) are being explored to compliment commonly used methods such as ELISA.

Why is there a need for new standards in this area, and what role is USP playing in the development of these new standards?

Dr. Kibbey: While many unique methods for measuring residual HCP and DNA have been developed and validated, companies still struggle with appropriate development and validation of assays that are suitable and will meet regulatory requirements.

The standards-setting work of the United States Pharmacopeial Convention (USP) can provide industry with best practices or validated procedures that enable companies to develop their own assays, or, in the latter case, verify that the USP procedure is suitable for their purpose. Through the efforts of its expert committees and expert panels, USP develops standards for the identity, strength, quality and purity of medicines and their ingredients, and for the identification of impurities.

USP’s documentary standards are published as monographs, general chapters, or General Notices in USP’s compendia—United States Pharmacopeia and the National Formulary (USP–NF). USP also develops Reference Standards—chemical and biologic materials—used by manufacturers to evaluate their own products against tests and procedures included in USP’s standards.

Currently, USP has an expert panel charged with making recommendations for a new general chapter numbered below 1000 that will include a validated test and associated reference standards for quantitation of residual E. coli and CHO genomic DNA in recombinant biotherapeutics. This proposed general chapter will be published in 2014 in Pharmacopeial Forum (PF), USP’s online, free-access journal for receiving comments on proposed or revised standards.

Another expert panel is developing a new informational general chapter (numbered above 1000) containing best practices for development and validation of residual HCP procedures. Unlike general chapters designated below 1000, those above 1000 do not include specific tests, system suitability criteria, or acceptance criteria.

As standards for residual impurities in biotech products are developed, USP will continue to solicit input from a wide range of stakeholders. This is accomplished through publication in PF of developing standards (where stakeholders may comment), as well as public meetings where industry and regulatory stakeholders have an opportunity to share their perspectives with USP.

On June 3–4, 2013, USP and the Biopharmaceutical Emerging Best Practices Association (BEBPA) will co-host a workshop entitled, “Measurement of Residual Host Cell Protein and DNA in Biotechnology Products.” The workshop will feature discussions on platform approaches for measurement of residual host-cell proteins; reagent quality issues for residual impurity assays; emerging technologies and best practices for measurement of host-cell proteins; and U.S. and European regulatory perspectives regarding these process-related impurities.

The workshop will also highlight USP’s ongoing work related to standards for residual host-cell impurities. Information on the workshop may be accessed at: http://uspgo.to/host-cell-protein.

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